Radiation-induced double-strand breaks (DSBs) are fundamental threats to genomic integrity that result in genomic instability if not properly repaired, which can in turn lead to cancer and cell death. Although we know a great deal about the pathways of DSB repair, we know very little about how DSB repair occurs in its natural context in the cell, that is, chromatin. Chromatin by its very nature is an impediment for proteins accessing the DNA, yet the repair machinery is somehow able to navigate through the chromatin and successfully repair DNA damage. Chromatin also plays a key role in transducing the cell's response to DNA damage via the DNA damage cell cycle checkpoint. Until recently, there has been a large gap in our understanding as to how the DNA damage checkpoint is turned off in order to allow cells to reenter the cell cycle and survive after DNA repair is complete. Integral to this process is the way that the cell senses that DNA repair is complete, which has also been a long-standing mystery. We have recently discovered that the restoration of the chromatin structure over the newly-repaired DNA, rather than DNA repair itself, is the elusive signal for inactivation, or recovery of the DNA damage checkpoint (Chen et al., Cell 2008) in order to allow cell survival after DSB repair. Although our studies have revealed a novel link between chromatin structure, checkpoint recovery and cell cycle re-entry after DNA repair, many questions remain to be answered. The proposed studies will uncover the elusive mechanism used by eukaryotic cells to turn off the DNA damage checkpoint after DNA repair is complete. By elucidating the mechanism whereby restoration of chromatin carrying this specific histone modification signals to the DNA damage checkpoint machinery that DNA repair is complete, we hope to fill significant gaps in our current knowledge of the chromosomal repair process. We will also identify novel proteins involved in turning off the DNA damage checkpoint that will be novel targets for therapeutic intervention in order to prevent inactivation of the damage checkpoint after irradiation of cancer cells, in order to prevent cancer cells from dividing.